The long-term objective of the proposed research is to create a fully automated hand-held platform for nanopore-based biomarker detection and quantitation. Our single-molecule assay currently has a time-to- results of 5 minutes or less, achieves pM sensitivity without optimization, and costs $2 (materials and labor) per test by using a disposable test strip model. Test results can be encrypted and reported to a user via the cloud, resulting in a platform with tremendous potential for affordable, point of care molecular diagnostics. In a nanopore device, voltage force captures individual DNA from a fluidic chamber above the pore, and the measured current through the pore temporarily shifts to signal the presence and passing of each molecule. To achieve detection of specific biomarkers from among a population of other molecules, our method uses bi- functional fusion molecules; one end of the fusion binds the target biomarker, while the other end anchors the fusion/biomarker complex to a unique location on a dsDNA scaffold. When each scaffold/fusion passes through the pore, the occupancy status of the fusion is detected because the event signature is distinct when the biomarker is bound, providing a simple yes/no criterion for every captured molecule. Our assays are also designed so that background-molecule events are distinguishable from scaffold/fusion events. Building on preliminary data, the objectives of this Phase I effort are: 1) to advance the bimolecular engineering steps and analysis methods to optimize biomarker detection performance, and 2) to demonstrate detection of three clinically relevant protein biomarkers: a bacterial coat protein, a human glycoprotein, and an anti-viral antibody. Aim 1. Create each dsDNA/fusion molecule reagent that will bind each target biomarker in solution. Aim 2. Perform nanopore experiments to detect the presence of DNA/fusion/protein complexes, and thereby signal the presence of each biomarker. Also, implement tools to assign statistical significance to detection. Aim 3. Titrate biomarker concentration and establish the limit of biomarker detection. Also, perform nanopore experiments in titered amounts of serum as background, to emulate a realistic diagnostic scenario.